ChIPonChip and Differential Location Analysis

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ChIP-on-Chip and Differential Location Analysis

• Junguk Hur
• School of Informatics

October 4, 2005
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Overview

• Introduction to Transcriptional Regulation
• ChIP-on-Chip (ChIP-Chip)
• Current Approaches
• Our Approach

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The Central Dogma
DNA
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Genes need to be regulated
If gene regulation goes awry? gt
Developmental abnormality
gt Diseases such as Chronic myeloid
leukemia rheumatoid arthritis
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• transcription
• post transcription (RNA stability)
• post transcription (translational control)
• post translation (not considered gene
  regulation)
• usually, when we speak of gene regulation, we are
  referring to transcriptional regulationthe
  transcriptome

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Transcriptional Regulation
DNA binding proteins
Non-coding region
Gene 1
Activator
Repressor
RNA transcript
Gene 2
Binding sites (specific sequences)
Gene 3
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Transcriptional Regulation
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Transcription Factor Binding Sites

• Gene regulatory proteins contain structural
  elements that can read DNA sequence motifs
• The amino acid DNA recognition is not
  straightforward
• Experiments can pinpoint binding sites on DNA

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Modeling Binding Sites
Given a set of (aligned) binding sites

• Consensus sequence
• Probabilistic model(profile of a binding site)

GCGGGGCCGGGC TGGGGGCGGGGT AGGGGGCGGGGG TAGGGGCCGGG
C TGGGGGCGGGGT TGGGGGCCGGGC ATGGGGCGGGGC GTGGGGCGG
GGC AAAGGGCCGGGC GGGAGGCCGGGA GCGGGGCGGGGC GAGGGGA
CGAGT CCGGGGCGGTCC ATGGGGCGGGGC
NNGGGGCNGGGC
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Overview

• Introduction to Transcriptional Regulation
• ChIP-on-Chip (ChIP-Chip)
• Current Approaches
• Our Approach

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ChIP-on-Chip

• Based on
• ChIP (Chromatin Immuno-Precipitation)
• Microarray
• In vivo assay
• Genome-wide location analysis

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Chromatin Immuno Precipitation (ChIP)

• Using antibody of a protein of interest
• DNA bound to specific protein are enriched.

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ChIP-on-Chip (Ren et al.)
Array of intergenic sequences from the whole
genome
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Protein Binding Microarray (PBM)(Bulyk et al.)

• In vitro assay
• DNA-binding protein of interest is expressed with
  an epitope tag, purified and then bound directly
  to a double-strand DNA microarray
• Can overcome the shortcomings of ChIP-on-Chip
• Poor enrichment
• No available antibody
• Unknown culture condition or time points

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Protein Binding Microarray
Whole-genome yeast intergenic microarray bound by
Rap1
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ChIP-on-Chip vs PBM

• Done by Mukherjee et al.
• Useful when ChIP-on-Chip does not result in
  enough enrichment
• Lee et al. , Lieb et al.

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Overview

• Introduction to Transcriptional Regulation
• ChIP-on-Chip (ChIP-Chip)
• Current Approaches
• Our Approach

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Approaches

• Representative TFBS (Motif) Discovery
• Understanding Regulatory Modules

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Motif Discovery

• MEME (Expectation Maximization)
• CONSENSUS (greedy multiple alignment)
• WINNOWER (Clique finding in graphs)
• SP-STAR (Sum of pairs scoring)
• MITRA (Mismatch trees to prune exhaustive search
  space)
• BioProspector (Gibbs Sampling Based)
• MDScan (Differential weight for sequences)
• Motif Regressor
• EBMF (Energy Based Motif Finding)

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Transcriptional regulatory codeby Harbison et al.

• Saccharomyces cerevisiae (budding yeast) -
  Eukaryote
• TFBS binding analysis
• Simple regulatory models
• 203 TFs in rich media 84 TFs in at least 1 in
  12 other environmental conditions
• Genome-wide location data 11,000 unique
  interaction (p lt 0.001)

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Transcriptional regulatory codeby Harbison et al.

• Identification of transcription factor binding
  site specificities

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Transcriptional regulatory codeby Harbison et al.

• Construction of regulatory map of Yeast

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Transcriptional regulatory code by Harbison et al.

• Promoter architectures

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Transcriptional regulatory codeby Harbison et al.

• Environment-specific use of regulatory codes

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Overview

• Introduction to Transcriptional Regulation
• ChIP-on-Chip (ChIP-Chip)
• Current Approaches
• Our Approach

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Our Approaches

• Better understanding of differential binding of
  TF and DNA in different conditions by using
  ChIP-on-Chip and gene expression data.

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Obstacles in TFBS Analysis

• Variation in binding sequences might be
  problematic in motif discovery process.
• But for differential binding, there is no
  sequence discrepancy.
• For eukaryotic systems, lots of transcription
  factors (TFs) work together with other TFs
  affecting each others binding to DNA

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Causes of Differential Binding

• We suspect the possible causes for this
  differential binding to be
• Changes in the TF expression
• Changes in other TFs expression
• Modifications in TFs (protein level)
• Changes in physical structures (epigenetic
  features)
• Other unknown reasons

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Cooperations in TFs
Condition 1
Condition 2
Condition 3

• What has caused the difference in the binding
  affinity?

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Differentially Bound Promoters

• Simple correlation
• (A, B binding ratio of TF
  in condition 1 and 2 respectively)

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Differentially Bound Promoters

• How can we confirm which other TF(s) is involved?

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Methods

• How can we confirm which other TF(s) is involved?
• Sequence analyses on the differentially bound
  promoters?
• Comparison of ChIP-on-Chip results?
• Protein-protein interaction between TFs?
• Other possible analysis
• Gene Ontology distribution of differentially
  bound promoters

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Expected Results

• We may be able to use heterogeneous experimental
  data to reveal the underlying mechanisms of
  differential binding of transcription factor to
  cis-regulatory region.

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• Thank you
• Any question and suggestion ?